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HEPES has been described as one of the best all-purpose buffers available for biological research. At biological pH, the molecule is zwitterionic, and is effective as a buffer at pH 6.8 to 8.2 (pKa 7.55). It is typically used in cell culture at concentration between 5mM to 30 mM. HEPES has been used in a wide variety of applications, including tissue culture. It is commonly used to buffer cell culture media in air. HEPES finds its usage in in vitro experiments on Mg.

White crystalline powder

Biological buffer.

Non-toxic to cell. It is used as a hydrogen ion buffer, which can control the constant pH range for long term. The concentration is 10-50mmol/L. Generally, 20mmol/LHEPES in nutrient solution can get buffer ability.

ChEBI: A HEPES that is ethanesulfonate substituted by a 4-(2-hydroxyethyl)piperazin-4-ium-1-yl group at position 2.

Multi purpose buffer used in biological research.

HEPES serves as a buffering agent and transfection reagent that mediates cytoplasmic delivery of proteins into cells, and mixing it with proteins such as antibodies, recombinant proteins, and small peptides yields protein–HEPES mixtures suitable for transfection into cultured cells[1]. As a zwitterionic buffering agent in cell culture media, HEPES activates MiT/TFE-dependent lysosomal-autophagic gene networks and induces lysosome biogenesis, while its addition to culture media also triggers a lysosomal stress-related inflammatory phenotype and affects host-immune gene programs in RAW macrophages[2]. HEPES serves as a building block for molecular engineering of biomedical polymers, functioning as a pendant group in the zwitterionic polymer polyHEPES-pz intended for biomedical applications[3].

The general procedure for the synthesis of 4-hydroxyethylpiperazineethanesulfonic acid from N-hydroxyethylpiperazine and sodium 2-hydroxyethylsulfonate is as follows: 0.5 mole of N-hydroxyethylpiperazine, 0.565 mole of sodium 2-hydroxyethylsulfonate, and 500 ml of methanol were added to a 1000 ml four-necked flask fitted with a refluxing condenser tube, and the condensation reaction was carried out under sufficient stirring. The reaction was first carried out at 50 °C for 2.5 minutes, then gradually heated to reflux and continued for 3.0 hours. The reaction was cooled after completion of the reaction to give a reaction mixture containing 4-hydroxyethylpiperazine ethanesulfonate. The yield of 4-hydroxyethylpiperazine ethanesulfonate was 93.6% as analyzed by high performance liquid chromatography. A 20 liter solution of 4-hydroxyethylpiperazine ethanesulfonate at a mass concentration of 15 wt% was diluted to 5 wt% with deionized water under stirring, followed by removal of sodium chloride by dialysis. Then the purified HEPES product was obtained by evaporation concentration, cooling crystallization and filtration. Finally, the product was washed once with anhydrous methanol and dried under vacuum to obtain purified HEPES.A sample of purified HEPES was dissolved and tested for purity, which showed a purity of 99.9%, a yield of 85.6%, a pH of 6.46, and a concentration of chloride ions of 6.8 μg/g.

Room temperature

Crystallise the acid from hot EtOH and water. It is a useful buffer. [Beilstein 23 V 376.]

[1] Patent: CN106905262, 2017, A. Location in patent: Paragraph 0034; 0035; 0047